Biological laser printing via indirect photon-biomaterial interactions

ABSTRACT

A method of laser forward transfer is disclosed. Photon energy is directed through a photon-transparent support and absorbed by a polymer interlayer coated thereon. The energized interlayer causes the transfer of a biological material coated thereon across a gap and onto a receiving substrate.

This application is a continuation application of U.S. patentapplication Ser. No. 10/863,850, filed on Jun. 4, 2004 and allowed,which claims the benefit of U.S. Provisional Patent Application No.60/476,377, filed on Jun. 6, 2003 and U.S. Provisional PatentApplication No. 60/542,841, filed on Feb. 10, 2004. All applicationsnamed are incorporated by reference. U.S. patent application Ser. No.10/863,833, filed on Jun. 4, 2004 is also incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is generally related to methods of laser and/or photontransfer.

2. Description of the Prior Art

Direct write technologies have gained popularity with the increasedinterest in biological sensors and microarrays, and the push forengineered tissues to replace organ transplants. These techniques allowfor increased ability to manipulate biological materials in very smallvolumes with much better accuracy than has been previously possible.Some of the most promising techniques for use in controlling andtransferring biological materials are matrix assisted pulsed laserevaporation direct write (MAPLE DW, see U.S. Pat. No. 6,177,151 toChrisey et al., (all referenced patents, patent applications andpublications are incorporated herein by reference), dip-pennanolithography (DPN), scanning probe microscopy (SPM), microcontactprinting (MCP), and laser guidance direct write.

MAPLE DW focuses a pulsed laser source at the interface of a quartz“ribbon” (analogous to a typewriter, it is usually a quartz slide with acoating containing a mixture of a matrix support material and thebiological materials of interest) to cause the ablation of a smallamount of the interfacial matrix material, which then causes theremaining bulk matrix and biological material to be expelled from theribbon in a bubble-jetting effect. The expelled material travelsthrough-space, away from the laser and ribbon to a receiving substrate.This results in a spot of transferred material approximately 100 μm indiameter with pL scale volumes. MAPLE DW has limited applications orinherent limitations due to the physical properties of the biologicalmaterials and surrounding media needed to ensure accurate patternformation. Specifically, MAPLE DW requires that a mixture of transfermaterial and a matrix material be presented to the laser source. Thematrix must be of higher volatility than the transfer material andstrongly absorb the incident radiation. In addition, the reproducibilityof the technique can be low due to inconsistencies in the parametersnecessary for ablation of the matrix material. Also, because theabsorptivity of certain matrix materials is quite low, there is thepotential for damage to biological materials from direct and indirectinteraction with the incident laser radiation.

DPN and SPM are direct-write techniques that offer the possibility ofwriting very small amounts of material. DPN has been used to write linesof collagen 30-50 nm in width. Both these techniques have been used tolay down patterns of organic adhesion molecules, which are then used foradhesion of biological molecules of interest. This leads to difficultyin patterning multiple biological material types and difficulty in rapiddesign of micro-scale constructs.

MCP techniques utilize biomaterial coated polymer stamps to transferbiomaterials to more adhesive substrates. It is possible to obtainsub-micron features using MCP, but there is little control over theamount of material transferred. It is possible that different materialscould be patterned using multiple stampings, but this could lead topossible contamination of stamps and has inherent limitations in theproximity of the different material types. Also, MCP is dependent onbiomaterial-substrate adhesion, and therefore not universal to allsubstrate materials.

Laser-guided DW uses a “loose” form of optical trapping to guide cellsalong the beam axis and to a receiving substrate. The use of a low NAlens allows for a continuous stream of cells or molecules to betransferred to a target substrate. This technique allows formanipulation and placement of individual cells, but has limitations inthe fact that it can take hours to write a moderate number of cells. Inaddition there is the potential for DNA damage from the extended timethat cells or other biological materials are exposed to the laser beam.

Laser transfer of biological materials presents a challenge due to thefragility of many biological materials. They can be harmed by shearstress when they are removed from the target substrate and by impactstress when they land on the receiving substrate. DNA in particular canbe uncoiled by such stresses. Heat can denature many biologicalmaterials. UV damage can also result when a UV laser is used.

In some other techniques, the target substrate is coated with severallayers of materials. The outermost layer, that is, the layer closest tothe receiving substrate, consists of the material to be deposited andthe innermost layer consists of a material that absorbs laser energy andbecomes vaporized, causing the outermost layer to be propelled againstthe receiving substrate. Variations of this technique are described in,for example, the following U.S. patents and publications incorporatedherein by reference: U.S. Pat. No. 5,308,737 to Bills et al., U.S. Pat.No. 5,171,650 to Ellis et al., U.S. Pat. No. 5,256,506 to Ellis et al.,U.S. Pat. No. 4,987,006 to Williams et al., U.S. Pat. No. 5,156,938 toFoley et al. and Tolbert et al., “Laser Ablation Transfer Imaging UsingPicosecond Optical pulses: Ultra-High Speed, Lower Threshold and HighResolution” Journal of Imaging Science and Technology, Vol. 37, No. 5,Sep./Oct. 1993 pp. 485-489. A disadvantage of these methods is that theycan still expose the material to be deposited to high amounts of energythat may not be suitable for biological materials.

SUMMARY OF THE INVENTION

The invention comprises a method for printing materials comprising thesteps of: providing a receiving substrate, providing a target substrate,providing a source of photon energy, and directing the photon energy.The target substrate comprises a photon-transparent support, aphoton-absorbent interlayer comprising a polymer coated on the support,and a transfer material comprising a biological material coated on topof the interlayer opposite to the support. The photon energy is directedthrough the transparent support so that it strikes the interlayer. Aportion of the interlayer is energized by absorption of the photonenergy, which causes a transfer of a portion of the transfer materialacross a gap between the target substrate and the receiving substrateand onto the receiving substrate

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates an apparatus used to perform the methodof the invention.

FIG. 2 shows a micrograph of an interlayer that was not removed by alaser pulse.

FIG. 3 shows a micrograph of an interlayer that was ablated by a laserpulse.

FIG. 4 shows a micrograph of transferred cells.

FIG. 5 shows micrographs of and schematically illustrates a seededthree-layer scaffold.

FIG. 6 shows a micrograph of transferred spots of BSA solution.

FIG. 7 shows a fluorescence micrograph of a large-scale array oftransferred human osteosarcoma cells.

FIG. 8 shows a micrograph of transferred spots of BSA solution and thetopology of one spot.

FIG. 9 shows a micrograph of a BSA microarray.

FIG. 10 shows a graph of calculated temperature versus time for atitanium interlayer/biolayer interface and for the middle of a 10 μmaqueous biolayer following a 30 ns laser pulse.

FIG. 11 shows a graph of calculated thermal penetration into a 10 μmaqueous biolayer.

FIG. 12 shows fluorescence micrographs of human osteosarcoma cells andmouse endothelial cells.

DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS

The invention provides a method for transfer of biological materialsthat can allow for control of material placement and volume, and canprevent damage to biological structure and function. These criteria arerelevant to designing new processes and techniques for tissueengineering, microfluidic cell and protein-based microsensors, genomicand proteomic microarrays and nanoarrays, biological or chemicalsensors, and cell-specific culturing applications.

The invention uses photon energy, such as a laser, incident upon aninterlayer to induce a biological material to be forward-transferredthrough space to a receiving substrate, microns to millimeters away fromthe target support. This eliminates the requirement of a laser-matrixinteraction as in MAPLE DW. The interlayer converts photon energy intothermal energy. Laser absorption and energy conversion by the interlayerresults in the removal of a three dimensional pixel of biomaterial fromthe target though one of a number of mechanisms, and propelling analiquot of biomaterial through air towards a receiving substrate. Theamount of biomaterial transferred in a single pixel is a function ofseveral factors including the laser spot size, the thickness of thebiomaterial layer on the ribbon, and the laser fluence. The method isindependent of the type of biological material present. Materialdeposition may be done at ambient temperatures under room conditions.The technique allows for transfer of very small transferred volumes (<pLscale), spot sizes as small as 20-50 μm, and has the demonstratedability to deposit patterns of single cells.

The method can have several key advantages over MAPLE DW. Laserinteraction with the transfer layer is indirect. This means thatvirtually any material in a wet or dry condition can be transferredwithout having to consider its ability to absorb incoming radiation,which is an imperative requirement for a successful MAPLE DW transfer.This also means that laser absorption can be essentially constant acrossmaterials and therefore, accurate transfers can be easier andreproducibility can be greatly increased. Further, interaction of thelaser with the transferred biological material is substantiallyeliminated. This reduces the possibility of genetic or biological damagefrom the relatively high energies of the focused laser beam. As thebiological layer also acts as the laser absorption layer in MAPLE DW,transfer conditions can vary greatly due to changes in the biologicallayer, altering the laser adsorption properties that are central toachieving the successful transfer of material. Calculations show thatmuch of the laser energy (>99%) passes completely through the liquid,raising the possibility that the UV laser light could damage thebiological material (Parkinson et al., “Absorption cross-sectionmeasurements of water vapor in the wavelength region 181-199 nm,” Chem.Phys., 204(31), 31 (2003)). Single and double strand comet assays haveshown little to no observable genetic damage to the cells, but UVexposure could still hinder other aspects of biological activity viaradical formation and cellular response to the UV. Finally, controllingthe MAPLE DW transfer process for biological materials is moredifficult, due to their poor UV absorption properties (aqueous mediums).In essence, the laser is absorbed by and could cause vaporizationthrough the entire volume of material with which it interacts. This canlead to problems in attaining reproducible results.

The technique allows for the stepwise writing of biological materialswithout the use of masks or patterning techniques. It can have theability to write materials in a three-dimensional manner(layer-by-layer) and to write onto non-planar surfaces. Because of thetransfer mechanism, it is possible to transfer virtually any biologicalmaterial, independent of physical properties such as phase, viscosity,homogeneity, light absorbance, and volatility. This method also canallow for rapidly transferring a large number of cells of the same typeor a variety of types in any order and in close proximity. With nodirect contact with the receiving substrate, there is less concern aboutcontamination and with the relatively small transfer volumes, it ispossible to obtain very accurate transfer placement.

FIG. 1 schematically illustrates an apparatus 10 that may be used toperform the method of the invention. The apparatus 10 includes a targetsubstrate 12, which comprises a photon-transparent support 13 having aninterlayer 14 coated thereon, and a transfer material 16 coated on theinterlayer. The transfer material comprises a biological material. Apulsed laser 18 sends laser pulses 19 into the target substrate andthrough the support. The pulses are absorbed by the interlayer, whichcauses a portion 20 of the transfer material to be transferred to areceiving substrate 22. The target substrate, the receiving substrate,and the photon energy source can be moveable with respect to each other.

Control of the beam can be done via an optical stage 24 and manipulationof the target and receiving substrates can be accomplished bycomputer-controlled stages 26 and CAD/CAM programs that allow for designof complex shapes, and patterns and the deposition of differentmaterials in a precise and ordered manner. Independent manipulation ofribbon and substrate can be done via a computer-controlled stage.

The source of photon energy may be any photon source that providessufficient photons to cause the transfer. A suitable photon source is alaser, such as a continuous laser or a pulsed laser. The fluence of apulsed laser should be chosen such that the transfer is accomplishedwithout causing any undesirable damage to the biological material, withor without removal of the interlayer. Pulsed lasers are commerciallyavailable within the full spectral range from UV to IR. Typically, suchlasers emit light having a wavelength in the range of about 157-10600nm, a pulsewidth of about 10⁻¹²-10⁻⁶ second and a pulse repetitionfrequency of about 0 to greater than 100,000 Hz. Examples of suitablelasers include, but are not limited to, excimer lasers operating at 193and 248 nm and frequency quadrupled or tripled Nd:YAG laser operating at266 and 355 nm. Suitable ranges of fluence include, but are not limitedto, from about 1 to about 1000 mJ/cm², at least about 0.1 mJ/cm², and atleast about 1 nJ/cm². A 193 nm Lambda Physik 300 ArF excimer laser or afrequency tripled Nd:YAG laser (355 nm) may be used for transfers. Thedimensions of the laser energy can be controlled by any means known inthe art so that only a precisely defined area of the target substrate isexposed to the laser energy and so that only a precisely defined portionof the transfer material is exposed. The laser energy can be focusedthrough an objective or lens to narrow the beam and expose a smallerportion of transfer material. The beam can be focused down to anapproximately 10-150 μm spot size at the ribbon interlayer. Thisincreases the possible resolution of the deposited biological material.Other suitable photon sources include, but are not limited to, a flashlamp and a maser. A flash lamp may be more divergent and require opticsto force the light to propagate parallel/co-linear.

The photon-transparent support can be made of any material that issubstantially transparent to the particular photons that make up thephoton energy. Suitable support materials include, but are not limitedto, quartz, a glass, a salt, and a polymer. Quartz is suitable when UVphotons are used.

The interlayer can be made of any material that significantly absorbsthe photon energy, and may be inorganic. Suitable interlayer materialsinclude, but are not limited to, a metal, a metal oxide, titanium metal,titanium dioxide, chrome, molybdenum, gold, and a polymer, and maycomprise more than one layer. Suitable ranges of thickness of theinterlayer include, but are not limited to, 10 Å to 10 μm and 5 to 100nm.

The transfer material can be coated on the support by numeroustechniques, including but not limited to spin coating, spray coating,dipping, doctor blading, roller coating, and screen-printing, and may bein the form of a powder, solid, liquid or gel. The surface of the targetsubstrate can be broken into separate regions with different transfermaterials in order to be able to deposit different biological materialswithout having to change the target substrate.

The biological material can be any material of a biological nature,whether naturally occurring or engineered, or synthetic equivalentsthereof, include, but are not limited to, proteins, hormones, enzymes,antibodies, DNA, RNA, nucleic acids, aptamers, antigens, lipids,oligopeptides, polypeptides, cofactors, polysaccharides, andbiocompatible materials such as tissue scaffolding material, ceramic, orpolymer. The biological material can be cells, living or non-living. Thecells may be stained cells and may be prokaryotic or eukaryotic. Livingcells may remain living on the receiving substrate after the transfer iscomplete, or the cells may be dead. The cells may be dried orlyophilized. The cells may also be in a solution that is evaporatedduring the transfer, which may also be used as a method of drying cells,although survival rates may be lower. This evaporation or drying may notbe from the heated interlayer but from the air currents during transitfrom the target to the receiving substrate. A cushioning coating on thereceiving substrate may help the cells to survive the impact with thereceiving substrate. Suitable cushioning coatings include, but are notlimited to, hydrogel and polymers, which may be at least 10 μm thick,and aqueous solutions. There can be minimal to no DNA strand breaks inthe transferred cells. The receiving substrate may be a microtitreplate, or may be a living substrate such as an animal or plant.

In some embodiments, the transfer material also comprises a biomatrixalong with the biological material. The biomatrix can be any materialthat is compatible with the biological material and that does notprevent the transfer. The biomatrix may contribute to sustaining thebiological activity of the biological material. The biological materialand the biomatrix may mixed together or in separate layers. Suitablebiomatrices include, but are not limited to, cell medium, water,glycerol, a mixture of water and glycerol, mammalian serums, bufferedsalt solution, biocompatible polymers, extracellular matrix, organictissue scaffolding, inorganic tissue scaffolding, biocompatiblesurfactants, Tween, sodium dodecyl sulfate, cell medium, cell nutrient,natural hydrogel, synthetic hydrogel, surfactant, antibiotic, antibody,antigen, dimethylsulfoxide, water/dimethylsulfoxide mixture, agarose,saline solution, dielectric particles, metal particles, aqueousinorganic salt solution, nitrocellulose gel, sol gel, ceramic composite,DNA-coated particles or nanoparticles, and RNA-coated particles ornanoparticles.

In some embodiments, the interlayer will remain substantially intact andadhered to the substrate when it absorbs the laser energy. A micrographillustrating this effect is shown in FIG. 2. The titanium interlayerremained intact, although with mild damage due to rapid heating andcooling. In this case, the mechanism of transfer may be, but is notlimited to, photomechanical and/or photothermal shock. This effect maybe useful when the biological material to be transferred may be damagedby exposure to higher levels of energy. The laser penetration into theinterlayer may be significantly less than the overall layer thickness.Models show that the temperature gain in the interlayer may be about100-1000 K, but that less than 5% of the transfer material reachestemperatures greater than ambient.

A portion of the biomatrix adjacent to the energized interlayer may beevaporated to cause the transfer. In this case the laser energy isabsorbed by the interlayer and rapidly converted into thermal energy.The superheated interlayer then causes flash ablation of a thin,interfacial portion of the biological support layer, which in turnforces jettison of the bulk biological materials. The transfer layerconsists of a mixture of the biomaterial and supporting biomatrix.

In some other embodiments, the interlayer may be removed or ablated fromthe target substrate when it absorbs the photon energy. A micrographillustrating this effect is shown in FIG. 3. Evidence of explosiveremoval of the titanium layer can be seen around the edges of thelaser-interlayer interaction region. However, this mechanism may beuseful when larger portions of a biological material, such as certainentire cells, are transferred. Some cells may be robust enough towithstand such higher levels of energy from the ablated interlayer orfrom direct exposure to the photon energy. Either mechanism may beappropriate when cells are transferred, depending on the type of cell.

The process can be repeated, exposing additional spots on the targetsubstrate, so that multiple deposits of biological material are formedon the receiving substrate. Multiple target substrates comprisingdifferent biological materials may also be used. These deposits may forma pattern, which can have three-dimensional layers. The pattern may havedesired properties or perform a desired function, including, but are notlimited to, a 2D or 3D cell construct, cell separation, cell isolation,and cell selection. The method may be capable of forming mesoscopicpatterns of cells in three-dimensional scaffolds through alayer-by-layer seeding approach.

The invention may improve upon previous cell deposition techniques interms of accuracy and speed, two components integral to the potentialuse of cell printing for engineering tissue. While previous work usingcell aggregates has, in essence, tried to straddle both traditionaltissue engineering methods and the stepwise approach, some studies sofar using the invention are focused on the ability to transfer specifictypes of cells or molecules to specific layers of model tissuescaffolds.

The invention can be capable of building biomaterial constructs in threedimensions through a layer-by-layer approach. This ability separates itfrom many previous techniques, such as soft lithography, that areexcellent for patterning single layers of cells, but are unable to buildupon initially deposited layers. With precise deposition techniques, itis possible to build three-dimensional, heterogeneous cell constructswhile incorporating growth factors, cytokines and other components,leading to an enhanced framework for tissue growth than traditional,scaffold-based tissues.

For both cell assaying and tissue engineering applications, it is may benecessary to not only deposit large numbers of cells over relativelylarge (mm) distances, but also to deposit multiple cell types in closeproximity to each other. Heterogeneous structures, such as tissues forregenerative medicine applications, are a perfect example of the need todeposit various cell types adjacent to one another. Further applicationsfor adjacent depositions of multiple types of materials include theability to transfer colonies of stem cells with various other types of“seed” cells, the deposition of growth factors with cells, and improvedcell separation methods.

Very few current technologies are able to transition between multiplecell types with efficiency. Applying the invention to producemulti-element arrays can be accomplished by tuning via changes in laserparameters, along with the design of targets with multiple “wells” orseparated areas for different types of biological materials. Theinvention is somewhat unique in that the target can be comprised ofmultiple wells, each containing a unique cell type or biomaterial.Targets that are 400 mm² and have as many as 36 1×1 mm² wells have beendesigned. This allows for rapid deposition of multiple cell types withno cross-contamination of the various biomaterials (no orifices) ordecrease in process efficiency (no need to change heads, lay downadditional adhesion layers, or go through different wash cycles, etc).Further, as the system is optically based, it is also possible to usethe technique to observe and select certain cell types. The ability toscan a sample and selectively isolate and/or deposit certain cells isvery attractive for cell separation experiments and genomics studies.

For protein solutions, the invention can be an alternative to arrayersthat use a solid pin or capillary-based fluidics. Protein solutions aretypically highly viscous, being prepared in 30-60% glycerol, arestrongly adherent to a variety of surfaces and tend to agglomerate athigh concentrations. These solutions tend to lead to agglomerationissues with solid pin arraying technologies and clogging and air bubbleformation in capillary fluidics with inkjet and quill-pin technologies.Using the present invention, the minimum spot diameter and volume perprinted droplet can be, but is not limited to, less at 30 microns and˜500 fL, respectively. The technique may require less than 500 mL ofstarting material, requires no washing cycles, and deposits spots ofprotein in a non-contact manner, which can eliminate potentialcontamination issues.

Models suggest that the laser absorption layer acts as an energyconversion material, converting the radiant laser energy via absorptionand conduction into heat, which then flows through the remainingabsorption layer to the interface with the protein solution.Calculations of laser penetration into the laser absorption materialshow that it is significantly less than the total layer thickness,thereby eliminating potential damage to proteins by incident UVradiation. Transient heat conduction models show temperature gains inthe laser absorption layer of 1400 K under typical laser and materialconditions. The effects of thermal stress upon the solid materials,their spatial displacements and the formation of thermal shock waves areunder investigation.

When using nanosecond laser pulses, thermal forces may drive theprinting mechanism. The ejection of protein solution away from thetarget may occur as a result of heat transfer through the absorptioninterlayer and subsequent vaporization of a portion of the proteinsolution in direct contact with the heated layer. The calculationsdiscussed above support this prediction, as the absorption layertemperatures are raised above the vaporization temperature for proteinsolutions. Alternatively, by utilizing near-ultrafast laser pulses,laser forward transfer experiments may be driven by mechanical forces,namely shock waves.

To measure the risk of UV exposure to the biomaterial during theprinting process, the percentage of UV laser energy that is absorbed inthe metal and metal oxide interlayer film was calculated. Biomaterialtransfer is achieved by focusing the laser at the interface of the laserabsorption layer and the optically transparent support layer. Theexponential absorption coefficient is based upon the calculatedpenetration depth (skin depth) for the metal. The calculated skin depthsat 266 nm for the two metals used as targets are given in Table 1. TABLE1 Interlayer Interlayer Conductivity material thickness (σ) (Ω⁻¹ · m⁻¹)Skin Depth (δ) (nm) Au 35 nm 4.5e7 2.2 Ti 75 nm 2.4e6 9.7 TiO₂ 85 nm —10.5¹¹“Skin Depth” for TiO₂ calculated from δ = 1/α, α = 4 · π · k/λ, k = 2(average of k⊥ and k∥)

The metal layer thickness used on the targets is much greater than theskin depths. Thus, greater than 99.9% of the non-rellected incidentlaser energy is absorbed by the solid metal prior to arriving at theabsorption layer-biomaterial interface. The TiO₂ layer also absorbs theradiation at 266 nm as indicated by the estimated “skin depth” (δ≡1/α)calculated from the complex index of refraction (n+ik) and theabsorption coefficient (α≡(4·πk)/λ). Using this calculation, it isestimated that greater than 99.9% of the incident laser energy is alsoabsorbed by the TiO₂ absorption layer prior to any interaction with thebiomaterial.

In order to understand the role of heat transfer during the process, theenergy transfer from the laser to the laser absorption layer was modeledby the time-dependent heat-transfer equation (parabolic formulation)with a source function given by the Lambert-Beer Law. The compositematerial heat-conduction equation was solved with a finite-elementsoftware package (FlexPDE). The models suggest that the laser absorptionlayer acts as an energy conversion material, converting the radiantlaser energy via absorption and conduction into heat, which then flowsthrough the remaining absorption layer to the interface with thebiolayer. The process may be a completely interfacial phenomenon.Calculations of laser penetration into the laser absorption materialshow that it can be significantly less than the total layer thickness.Transient heat conduction models of laser irradiation of the laserabsorption layer show temperature gains in the laser absorption layerfrom 150 to 450 K, increasing with laser fluence (FIG. 10). Heattransfer through the laser absorption layer showed little dependence onabsorption material thickness, as long as the thickness of theabsorption layer was greater than the penetration depth (skin depth) ofthe laser. The effects of thermal stress upon the solid materials, theirspatial displacements and the formation of thermal shock waves are underinvestigation.

In contrast to the rapid energy conversion and transfer seen in thelaser absorption layer, thermal penetration into the much thickeraqueous biomaterial layer (10 to 100 μm thick) is observed to benegligible. FIG. 11 shows the thermal penetration into the biolayer as afunction of distance at 50 and 500 ns after laser irradiation. As can beseen in the graph, at 50 ns post-irradiation, thermal penetration iscalculated to be less than 400 nm into the biolayer. Even 500 nspost-irradiation, the penetration is only 1.2 μm into the biolayer. Thishas been observed experimentally as well, when frozen crystals have beenobserved to be printed onto a room temperature substrate from a frozentarget support. Further, this model is an upper limit for thermalpenetration, as it uses an insulation model that does not allow forthermal cooling. In addition, the model does not account for thevaporization of the material that would occur as soon as the biomaterialat the interface reaches 400 K. Vaporization at the interface willreduce the amount of thermal penetration by acting as an insulationlayer and by reducing the ambient heat via heat of vaporization. Whilethe current model predicts that the first 5% of the material nearest theinterlayer is affected, it is likely that the amount of materialactually affected is much less. Therefore, it is predicted that fluidsprinted by this technique will incur little heating, minimizing thepotential for denaturing proteins during the printing process. Thetransfer of the solution may exhibit jetting phenomena as disclosed inU.S. patent application Ser. No. 10/237,072 to Young et al., filed Sep.9, 2002 and incorporated herein by reference.

Having described the invention, the following examples are given toillustrate specific applications of the invention. These specificexamples are not intended to limit the scope of the invention describedin this application.

The apparatus was composed of an optical setup designed to direct apulsed laser (either a Continuum Mini-lite quadrupled Nd:YAG (266 nm,3-5 ns FWHM, E_(max)=4 mJ, rep rate=1-15 Hz) or MPB Technologies PSX-100Excimer Laser 248 nm, 2.5 ns FWHM, E_(max)=5 mJ, rep rate=0.1-100 Hz)onto a “target” where transfer of a protein solution occurs. The laserwas split by a ⅛ beam splitter to an energy meter (Molecutron Max500).The rest of the beam traveled to a UV reflective mirror and directeddown (-Z) to a 10× microscope objective (LMU-10X-UVR, OFR, Inc.). Themicroscope objective focused the laser onto the target, which held thebiomaterial to be transferred, and allowed for observation of thetransfer process via a confocally aligned CCD camera (Sony DFW-V500).The microscope objective was attached to a micrometer, enabling changesto the focus and observation of the receiving substrate. Proteinmicroarrays were formed by timing laser pulses in conjunction with themovement of the substrates by computer-controlled stages.

The target consisted of an optically transparent quartz disk that wascoated with a metal or metal oxide via standard ion assisted electronbeam processing techniques (Thin Films Research, Boston, Mass.). EitherAu (thickness=300 or 1000 Å) or Ti (750 Å) metals or TiO₂ (850 Å) wasused as a laser absorption material. To print patterns of proteins, a0.5 to 5 μL aliquot of protein solution was spread homogeneously on topof this laser absorption layer. Control of the thickness of thebiomaterial or protein solution on the target was achieved by usingeither SU-8 photoresist or 3M PTFE Film Tape #63 over the metal or metaloxide film to produce a 20-100 μm deep well. Well sizes varied from 1mm² to 4 cm², and targets have been fabricated that contained between 4and 36 wells. The coated target was then placed in the apparatus, wherecomputer control allowed for selective deposition of protein droplets.The multi-welled target allowed for deposition of multiple proteinsolutions without the need to remove, replace, or wash/rinse the target.

Receiving substrates used for cell depositions were standard glassslides 1″×3″ coated with 50-200 μm of commercial basement membrane gel(MATRIGEL™ matrix, BD Biosciences). The gel was deposited as a liquid onthe slide and mechanically bladed to the required thickness. Substratesfor protein transfers were silinated glass microscope slide used asacquired (BD Biosciences). Ribbon to substrate distances wereapproximately 500 μm for cell transfers and 250 μm for protein solutiontransfers.

The target and the receiving substrate were on independentcomputer-controlled XY translation stages (Aerotech ATS36210, AerotechATS15030) with maximum translation speeds of 200 and 75 mm/s,respectively. While deposition rates were currently limited by laserrepetition rates (100 or 15 Hz), these stages allowed a theoreticallimit of 22,500 transfers/minute with 100 μm diameter spots spaced 100μm apart. Significantly higher deposition rates are therefore possiblewith an alternative laser running in the near kHz range. The receivingsubstrate was on a manual Z translation stage (Newport) which allowedfor ½″ of vertical travel to adjust for varying types of receivingsubstrates.

Example 1

Deposition of eukaryotic cells in a large-scale array format—Humanosteosarcoma cells (ATCC #: CRL-1427, designation: MG-63) were culturedas per literature, trypsinized and placed in a deposition media composedof 50% (v/v) DMEM, 45% (v/v) Fetal Bovine Serum and 5% glycerol (v/v).Cell concentration in the deposition media was found to be 10⁶ cells/mL.12 μL of the deposition solution was spread across a 4 cm² area of thetarget and then placed in the apparatus for transfer. Arrays of cellwere deposited onto 1×3″ glass slides coated with a 200 μm thickMatrigel™ basement membrane matrix, immersed in DMEM and placed in anincubator 5 minutes after transfer. Twenty hours post-transfer, thecells were washed with 1×PBS and assayed via a live/deadviability/cytotoxicity kit (Molecular Probes, L-3224) (FIG. 7).

In this deposition, the laser spot was 100 μm in diameter at the laserabsorption interface and laser fluence was 160 mJ/cm². Deposition spotswere approximately 100 μm in diameter and spaced 600 Mm apart. Initialcell concentrations on the receiving substrate were approximately 3-10cells per deposition spot, which correlates with cell concentrations inthe target solution (10⁶ cells/mL) and the calculated volume of thematerial transferred (3-10 μL/spot). Live/dead assays show that cellviability was near 100% 20 hours post-deposition. Several cell lines(ATCC designations: MG-63, EOMA GFP, C2C12, P19) have been printed andshow near 100% viability when deposited with both laser systems (Nd:YAGand XeF excimer) and with several types of laser absorption layers(TiO₂, Ti, Au). For feasibility studies of the invention as an arrayingtechnology, homogeneous arrays of MG63 cells with overall array areas of20 mm by 20 mm and deposition rates of 100 spots/s have been deposited.

Cell viability was tested with a live/dead viability kit (L-3224,Molecular Probes) consisting of two dyes: live test by calcein AM (dye1, 10 μM, ex/em ˜495 nm/515 nm) and membrane exclusion test by ethidiumhomodimer-1 (dye 2, 10 μM, ex/em ˜495 nm/635 nm). Images of transferredcells were obtained with a Hitachi HV-C20M CCD camera attached to aNikon Optiphot-2 microscope with an epifluorescent attachment after 30min of incubation in the viability assay solution.

Example 2

ATCC designation MG 63 human osteosarcoma cells and C2C12 mouse myoblastcells—MG63 cells were initially obtained from ATCC (USA) and cultured in5% humidified CO₂ in air at 37° C. in Dulbecco's Modified Eagle Medium(DMEM) with high glucose, 10% (v/v) fetal bovine serum, and 1% (v/v)streptomycin. FIG. 4 shows a patterned row of cells transferred from atitanium-coated quartz support and deposited onto a quartz substratecoated with matrigel (50 μm). The apparatus was as described above. Byvarying laser spot size, the number of cells deposited could becontrolled. At ˜100 μm spot size and an energy of 0.35 μJ/pulse,near-single cell transfers were obtained, at cell transfer rate of 1cell/shot (±0.5). Laser fluence was 4.4 mJ/cm².

Example 3

ATCC designation MG 63 human osteosarcoma cells—Human osteosarcoma cellsalong with 10 μm fluorescent beads were transferred from atitanium-coated quartz support using the energy conversion method. Thecells and beads were in a 25% glycerol, 25% cell media (DMEM), and 50%aqueous bead (0.15 M NaCl, 0.05% Tween 20, 0.02% thimerosal) solution.Laser fluence was 160 mJ/cm² and spot sizes were 100 μm in diameter.

Example 4

Verification that cells are not stressed—Certain cells can express heatshock proteins (i.e. HSP60, HSP70, etc.) during and after exposure tovarious stressors including elevated temperature and shear conditions.Immunocytochemical staining experiments (primary antibody: mouseanti-HSP60/HSP70, secondary antibody: fluorescein anti-mouse) wereperformed using MG63 osteosarcoma cell exposed to elevated incubatortemperatures (45° C.) for one hour, normal incubation conditions, andcells printed by the present process. A low level of fluorescenceemanated from laser printed cells, similar to or lower than that fromthe negative control cells. This implies that laser-printed cells didnot experience significant enough levels of stress during the printingprocess to express proteins known to be markers for heat and shearstress.

Example 5

Multiple cell types—Human osteosarcoma cells (MG-63) were deposited inan array format on a basement membrane with 800 μm spacing betweenspots. An array of mouse endothelial cells (ATCC #: CRL-2587,designation: EOMA GFP) was then transferred to the same substrate, butoffset 400 μm. The substrate was incubated post-transfer for 24 hours,and then tagged with DAPI nuclear stain (ex: 369 nm, FIG. 12(a)). FIG.12(a) shows that both cell types have taken up the nuclear stain andshow strong blue fluorescence at 465 nm. The EOMA cells can be opticallydifferentiated from the osteosarcoma cells due to genetic modificationsthat enable the expression of green fluorescent protein. FIG. 12(b) is aUV micrograph showing fluorescence at 510 nm emanating from the GFPexpressing EOMA cells. Both cell types show expected growth as comparedto non-laser transferred controls. Cell concentrations of the MG-63 cellsolution on the target (1.5×10⁸ cells/mL) were higher than the EOMA cellsolution (4.0×10⁷ cells/mL), causing the notable size difference in thetransfer spots. This demonstrates the feasibility of using the inventionfor design of heterogeneous tissues or development of multi-elementwhole-cell based biosensors.

Example 6

Three-dimensional scaffold—An example of the method's ability to depositin three dimensions is shown in FIG. 5. A bottom layer of basementmembrane (50 μm thick) was spread onto a glass substrate and droplets ofMG-63 human osteosarcoma cells were deposited approximately 100-150 μmapart. The slide was removed, and a 75 μm thick layer of basementmembrane was spread onto the substrate, and another line of cells wasdeposited overtop and orthogonally to the first. A third layer of 75 umMatrigel was spread and a third layer of cells was transferred. Thecells were then incubated overnight and a live/dead assay was performed(FIG. 7). The cells transferred into the first layer of basementmembrane show adhesion and expansion at 24 hours post transfer, as wouldbe expected if they were growing in a 2-D culture vessel. It has beenobserved that the printing process produces enough forward translationalenergy that the cells will embed in the receiving gel. Because the firstbasement membrane layer was relatively thin, the deposited cells wereable to pass through this layer, adhere to the glass substrate beneath,and grow. In contrast to the lower layer, the cells deposited into thesecond and third layers of basement membrane show ball-like growth,common to sarcoma cell lines when the cells are embedded in a matrixwith no accessible adherent surface.

Example 7

Transfer of protein solution—The invention can be capable of depositingvolumes of material ranging from hundreds of femtoliters to nanoliters.This capability is demonstrated in FIG. 6 where incident laser energywas varied to change deposited spot size of an aqueous bovine serumalbumin (BSA) solution. The solution used in these experiments wascomposed of 250 ng/μL biotinylated BSA with 0.1% (v/v) sodium dodecylsulfate and 50% (v/v) glycerol. Volume calculations based onprofilometry measurements of deposited material on hydrophilic glassslides show that it is possible to obtain transfer volumes from 1.6 μL(FIG. 6(a)) to 230 μL using laser fluences of 95 mJ/cm² and 350 mJ/cm²,respectively (FIG. 6(b)). The ability to tune the volume of materialtransferred over two orders of magnitude may come from the flexibilityinherent in the system. By changing the laser energy, the laser focalspot size, and the thickness of the biological material beingtransferred, it is possible to induce large effects on the resultingprinting process.

Reproducibility is a major concern in the development of a biologicalprinting technique. MAPLE DW suffered from difficulty in producingspot-to-spot reproducibility over large areas. Transfer of large-scalearrays of aqueous solutions has shown that it is possible to attain spotdiameters with a standard deviation of 9.3% for the spot diameter(N=200, spot diameter=61+/−5.7). This level of error in reproducibilityis comparable to other research devices producing much larger spots andis slightly larger than those quoted for standard, commercial-gradepiezo-tip and solid pin arrayers. However, it should be possible toreduce the variation in spot size significantly.

Example 8

Transfer of variable amounts of protein solution—Experiments wereperformed to determine the adjustability of transfer volume and toquantify the minimal volume capable of being reproducibly transferred.The protein solution used for these experiments was a 1 μg/mL bovineserum albumin (BSA) in 40% (v/v) glycerol, 60% (v/v) phosphate buffersolution (PBS), and 0.5% (w/v) sodium dodecyl sulfate (SDS) to enhancespreading of the solution on the target support. FIG. 8(a) shows amicrograph of a BSA array deposited by increasing the laser fluence from20 to 50 mJ/cm (laser spot diameter=125 μm). Optical microscopy was usedto determine the resulting spot areas, and optical profilometry was usedto measure the volumes of the printed droplets. Under these laserparameters, droplets with average diameters from 30 to 125 μm wereprinted, increasing with fluence. Profilometry measurements determinedthat the transferred protein spots have a contact angle of 50. The glassslide used in this experiment was wiped clean with methanol, but notcleaned in an acid and/or base bath to expose a truly hydrophilicsurface. Therefore, the glass surface is most likely slightlyhydrophobic due to environmental and laboratory contaminants (residualalcohol cleaning solutions, dust, salts, etc.). The small contact anglemeasured by the profilometer is most likely induced by the SDSsurfactant present in the protein solution.

FIG. 8(b) shows a sample topology of one of the deposited droplets, witha calculated volume of 2 pL, a 60 μm diameter, and a contact angle of5°. Due to limitations of the optical profilometer, accuratemeasurements could not be made on the 30 μm diameter droplets shown inthe bottom row of FIG. 8(a). Based on the volumes calculated for thelarger spots, the volume for the μm diameter spots of approximately 500fL (assuming a similar contact angle) was extrapolated. The volumes ofthe largest droplets (125 μm diameter) were measured to be 25 pL. It isalso possible to use this technique to produce drops with volumes in thenanoliter range by increasing the energy and diameter of the focusedlaser.

This data demonstrates that under optimal transfer conditions, theinvention can have the ability to print aliquots of protein solutionwith 2.5 times smaller spot sizes and 200 times smaller volumes thanpiezo-tip printers, which are the commercial-grade arrayers with thesmallest demonstrated spot size and volumes. In addition, by simplychanging the laser energy, the invention can print a range of volumes ofnearly three orders of magnitude. This versatility should enable theinvention to generate more dense protein microarrays than commercialarrayers as well as gradient arrays with varying concentrations andprotein densities for more quantitative analysis.

Example 9

Large scale array—FIG. 9(a) is a fluorescent image of a N=266 spotlaser-deposited, biotinylated bovine serum albumin (BSA) array. Dropletsof 250 ng/μL biotinylated BSA were first deposited onto anitrocellulose-coated glass slide (FAST™ slides, Schleicher & Schuell,Keene, N.H.) by the biological laser printer. After blocking themembrane with casein, the slide was exposed to cy5-conjugatedstreptavidin, and results were then analyzed on a GENEPIX® 4000Bmicroarray scanner (Axon Instruments, Inc.). This laser deposition andfluorescent tagging experiment demonstrated that this approach is ableto accurately place biotin-labeled protein onto recipient substrates andassay the presence of bound protein contained in ˜3000 μm² areas. FIG.9(b) is a higher magnification image showing the reproducibility of thedeposition technique. Preliminary results also showed protein activityfor the printed enzyme.

The level of reproducibility shown in FIG. 9 is consistent with otherresearch prototype devices transferring larger volumes of proteinsolution. It should be possible to reduce spot-to-spot variationsignificantly by using more homogenized fluid films on the target andmore sophisticated optics and/or lasers to ensure shot-to-shot energyfluctuations are minimized.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is therefore to beunderstood that the claimed invention may be practiced otherwise than asspecifically described.

1. A method for printing materials comprising the steps of: providing areceiving substrate; providing a target substrate comprising aphoton-transparent support, a photon absorbent interlayer comprising apolymer coated on the support, and a transfer material comprising abiological material coated on top of the interlayer opposite to thesupport; providing a source of photon energy; and directing the photonenergy through the transparent support so that the photon energy strikesthe interlayer; wherein a portion of the interlayer is energized by anabsorption of the photon energy; and wherein the energized interlayercauses a transfer of a portion of the transfer material across a gapbetween the target substrate and the receiving substrate and onto thereceiving substrate.
 2. The method of claim 1, wherein the energizedinterlayer remains intact and adhered to the target substrate.
 3. Themethod of claim 1, wherein the energized interlayer is removed.
 4. Themethod of claim 1, wherein the transfer material further comprises abiomatrix; and wherein the transfer occurs through an evaporation of aportion of the biomatrix adjacent to the energized interlayer.
 5. Themethod of claim 1, wherein the transfer occurs through a photomechanicalshock.
 6. The method of claim 1, wherein the transfer occurs through aphotothermal shock.
 7. The method of claim 1, wherein the transferoccurs through an ablation of the interlayer.
 8. The method of claim 1,wherein the photon transparent support comprises quartz.
 9. The methodof claim 1, wherein the photon transparent support comprises a materialselected from the group consisting of a glass, a salt, and a polymer.10. The method of claim 1, wherein the interlayer is 10 Angstroms to 10microns thick.
 11. The method of claim 1, wherein the receivingsubstrate comprises a cushioning coating.
 12. The method of claim 1,wherein the biological material is selected from the group consisting ofeukaryotic cells and prokaryotic cells.
 13. The method of claim 1,wherein the biological material is selected from the group consisting ofproteins, hormones, enzymes, antibodies, DNA, RNA, nucleic acids,aptamers, antigens, lipids, oligopeptides, polypeptides, cofactors, andpolysaccharides.
 14. The method of claim 1, wherein the transfermaterial comprises a combination of the biological material and asupporting biomatrix.
 15. The method of claim 14, wherein the biomatrixis selected from the group consisting of cell medium, water, glycerol, amixture of water and glycerol, mammalian serums, buffered salt solution,biocompatible polymers, extracellular matrix, organic tissuescaffolding, inorganic tissue scaffolding, biocompatible surfactants,sodium dodecyl sulfate, cell medium, cell nutrient, natural hydrogel,synthetic hydrogel, surfactant, antibiotic, antibody, antigen,dimethylsulfoxide, water/dimethylsulfoxide mixture, agarose, salinesolution, dielectric particles, metal particles, aqueous inorganic saltsolution, nitrocellulose gel, sol gel, ceramic composite, DNA-coatedparticles or nanoparticles, and RNA-coated particles or nanoparticles.16. The method of claim 14, wherein the biological material is cells.17. The method of claim 16, wherein the cells are living.
 18. The methodof claim 17, wherein the living cells remain living after the transferof a portion of the transfer material to the receiving substrate. 19.The method of claim 1, wherein the photon energy source is a pulsedlaser.
 20. The method of claim 1, wherein the target substrate, thereceiving substrate, and the photon energy source are moveable withrespect to each other.
 21. The method of claim 1, wherein the step ofproviding a target substrate is repeated one or more times using one ormore additional target substrates comprising one or more differenttransfer materials.
 22. The method of claim 1, wherein the directingstep is repeated to produce a pattern of transfer material on thereceiving substrate.
 23. The method of claim 22, wherein the patterncomprises a plurality of different transfer materials.
 24. The method ofclaim 22, wherein the pattern comprises a plurality of three-dimensionallayers.